Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
  • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
  • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
  • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to a polynucleotide comprising a nucleic acid sequence according to SEQ ID NO: 1, 2 or 3 or its fragment or derivative, or a polynucleotide which hybridizes with the nucleic acid sequence according to SEQ ID NO: 1, 2 or 3.
• the present invention further relates to polypeptides encoded by the nucleotide sequence or fragment or derivative of the nucleotide sequence according to SEQ ID NO: 1, 2 or 3.
• the polynucleotides and polypeptides can be used as medicaments, vaccines or diagnostics, in particular for the treatment, prevention and diagnosis of HIV Infections.
• HIV human immunodeficiency virus
• Phylogenetic analyzes of the worldwide circulating HIV strains have a main group (M) with 10 different sequence subtypes (AJ) (Kostrikis et al 1995; Leitner and Albert, 1995; Gaywee et al. 1996; World Health Organization Network for HIV Isolation and Characterization, 1994), which show sequence variations of up to 24% in the coat protein and also identifies the viruses of the O group, which in some reading frames differ from the viruses of the M- Differentiate group (Loussert Ajaka et al. 1995; Myers et al. 1996; Sharp et al. 1995; Sharp et al. 1999).
• AJ sequence subtypes
• viruses of the non-B subtype are responsible for the vast majority of new HIV-1 infections worldwide.
• the viruses of subtype C play an outstanding role in terms of the total number of infected people and the widespread spread of ⁇ eu infections, especially in South America and Asia. Because of this, the characterization of viruses of subtype C has an outstanding priority for diagnostic, therapeutic or preventive purposes.
• sub-type C viruses quickly spread through drug trafficking to south, central, and even northwest China, causing the epidemic to spread further within China.
• IDUs In to a recently published national HIV molecular epidemiological investigation report, almost all people infected with subtype C viruses are IDUs, accounting for around 40% of all HIV-infected IDUs in China. This suggests that subtype C viruses are among the most important HIV-1 subtypes that are prevalent among IDUs in China (Shao et al. 1998, Shao et al. 1994).
• subtype C HI-1 viruses are concerned, only non-recombinant representatives and four A / C recombinants, all of which originate from Africa, South America or India, have been published (Luo et al. 1995; Gao et al. 1998 ; Lole et al. 1999).
• the data collected so far on subtype C viruses in China are limited to genetic subtyping of the env gene (Luo et al. 1995; Yu et al. 1997; Salminen et al. 1995).
• Vaccines have been used in several clinical trials to combat HIV infection.
• the disappointing results seen in clinical trials include repeatedly reported breakthroughs in the vaccine. This was mainly attributed to the extensive sequence variations between the administered coat proteins and the infectious input virus, which is in fact mainly due to an insufficient characterization of the virus population circulating in a specific geographical region. This resulted in the generation of humoral and, to a lesser extent, line-mediated immune responses to viral antigens, which were not relevant to the viruses circulating in the population of the test area.
• low-affinity antibodies directed specifically against the coat protein not only have no neutralizing properties, but also contribute to an increase in infection by means of a complement or Fc receptor.
• the selected antigens and administration systems proved to be extremely weak for the induction of the cell-mediated immune response.
• vaccine preparations should contain mixtures of representative antigens. There is thus a need for the isolation and characterization of viruses of the subtype C, in particular for the cloning of the coding region.
• FIG. 1 shows a representation of the phylogenetic relationship of the region encoding the env gene C2V3 of the clone 97cn54 to the representatives of the important subtypes of HIV-1 (M group).
• cn-con-c stands for the env consensus sequence of the HIV-1 strains of sub-type C, which are prevalent in China.
• the phylogenetic family tree was created using the "neighbor joining" method.
• the values at the nodes indicate the "bootstraps" in%, which the grouping on the right supports. Only “bootstrap” values that reach or exceed 70% are given.
• the brackets on the right represent the sequences of the most important subtypes of HIV-1, M group.
• FIG. 2 shows a representation of the RTF analysis (Recombinant Identification Program), version 1.3, of the entire gagpol coding area of 97cn54 (window size: 200, threshold value for statistical significance: 90%, dealing with gaps: STRJP).
• the positions of the open reading frames of gag and pol are shown by arrows in the diagram above.
• RTP analysis was based on background comparisons using reference sequences derived from selected virus strains that are the major subtypes of HIV-1. Standard representatives are marked by different colors as shown.
• the x-axis indicates the nucleotide positions along the sequence comparison.
• the y-axis indicates the similarity of 97cn54 to the listed sub-types.
• FIG. 3 shows a representation of the phylogenetic relationship of different regions within the reading frames of gagpol derived from 97cn54 with standard representatives of the most important subtypes of HIV-1 (M group).
• M group standard representatives of the most important subtypes of HIV-1
• Gray areas indicate clusters of the analyzed sequences either with reference strains derived from subtype C (A, C, E, G) or from subtype B (B, D, F).
• the values at the nodes indicate the "bootstrap” values in percent by which the cluster on the right was confirmed. Only “bootstrap” values of 70% or more are shown.
• FIG. 4 shows an illustration of the RTP analysis, version 1.3, of different regions of 97cn54 (window size: 200, threshold value for statistical significance: 90%, dealing with gaps: STRIP).
• the analysis included (A) a sequence region of 1500 bp in length from the start codon of the vif gene to the 5 'end of env including vif, vpr, the first exon of tat and rev, vpu and the first 200 bp of the env gene and (B) an approximately 700 bp fragment overlapping 300 bp from the 3 'end of env, which encompasses the complete nef gene and portions of the 3' LTR region.
• the positions of the start codons vpr, tat, vpu, env, nef and the 5 'end of the 3' LTR region are indicated by arrows at the top of the diagrams.
• the RIP Analysis was based on background comparisons using sequences derived from selected virus strains representing the major subtypes of HIV-1. The specified standard representatives are identified by different colors.
• the x-axis indicates the nucleotide positions along the sequence comparison.
• the y-axis indicates the similarity of 97cn54 to the listed sub-types.
• C) and (D) show RIP analyzes of sequences from two independent C isolates (xj24 and xj 158) from China that overlap the vpr and vpu genes including the first exon from tat.
• Figure 5 shows the analysis of a phylogenetic family tree.
• Phylogenetic pedigrees were created using the "neighbor joining" method based on (A) a 380 bp fragment that overlaps 150 bp from the 3 'end of the vpr gene to the end of the vpu reading frame, (B) the first 290 bp of the coding region from nef and (C) to the 320 bp at the 3 'end of the nef gene.
• the values at the nodes indicate the "bootstrap” values in percent, by means of which the cluster on the right was confirmed. Only “bootstrap” values of 70% or more are shown.
• the brackets on the right represent the most important subtype sequences of HIV-1, group M.
• Figure 6 is a schematic representation of the mosaic organization of the 97cn54 genome.
• FIG. 7 is a representation of the comparison between known and experimentally proven CTL epitopes of prototype B (HIV-I LA L) and the corresponding amino acid sequences of the polypeptides gag, pol and env of strain 97cn54 of subtype C.
• the functional domains in GAG pl7 matrix, p24 capsid, pl5 nucleocapsid and linker protein
• POL PR protease, RT reverse transcriptase, IN integrase
• ENV g ⁇ l20 outer glycoprotein, gp41 transmembrane protein
• Haplotype restrictions of the known CTL epitopes of HIV-I LAI are indicated on the left or right margin.
• the green bars indicate sequence identity between the known epitope and the corresponding sequence of subtype C, blue bars indicate 2 or less conservative mismatches.
• Red bars represent sequence areas derived from subtype C with more than 2 conservative mismatches or non- conservative substitutions compared to the corresponding LAI-derived epitope.
• FIG. 8 shows the complete coding nucleotide sequence of 97cn54 of HIV-1, subtype C (SEQ ID NO: 1), with the corresponding amino acids in the one-letter code. All 3 reading frames are given. The asterisks represent stop codons.
• FIG. 9 shows a representation of the result of the activities of cytotoxic T cells in spleen cells from BALB / c mice after intramuscular immunization with the indicated DNA plasmids.
• Lymphoid cells obtained 3 weeks after the primary immunization from 5 mice per group, were co-cultivated with AMQMLKETI (one-letter code) Gag peptide-loaded syngeneic P815 mastocytoma cells (irradiated with 20,000 rad).
• Controls included spleen cells from non-immunized mice stimulated with peptide-loaded P815 cells. Cyto toxic effector cell populations were harvested after 5 days of in vitro culture.
• the cytotoxic responses were read against A20 cells loaded with the nonameric peptide listed above or against unloaded A20 cells in a standard 51 Cr release test.
• the data shown represent the mean values from triple approaches in each case. The standard deviations determined were less than 15% measured on average.
• epitope or "antigenic determinant” as used below mean an immunologically determinant group of an antigen that is specifically recognized by an antibody.
• An epitope can comprise amino acids in spatial or discontinuous conformation and comprises at least 3, preferably at least 5, amino acids.
• An epitope can also comprise a single segment of a polypeptide chain comprising a continuous amino acid sequence.
• polynucleotide refers to a single- or double-stranded heteropolymer composed of nucleotide units of any length, which can be either ribo- or deoxyribonucleotides.
• the term also includes modified nucleotides.
• derivative refers to a nucleic acid that also encodes the polypeptide (s) encoded by another nucleotide sequence, although its nucleotide sequence differs from the other nucleotide sequence. In this sense, the term “derivative” also denotes equivalents of the other nucleotide sequence which are present due to the degeneration of the genetic code.
• derivative includes, for example, nucleic acids which code the same polypeptides as the nucleotide sequence according to SEQ ID NO: 1, 2 or 3 , but have a different nucleotide sequence, or nucleic acid fragments which encode the same polypeptide as nucleic acid fragments of the nucleotide sequence according to SEQ ID NO: 1, 2 or 3 are also included.
• polypeptide refers to a chain of at least 2 amino acid residues linked together by peptide bonds. The term therefore includes all amino acid chains, e.g. Ohgopeptides and proteins. The term also refers to those amino acid chains in which one or more amino acids are modified, e.g. by acetylation, glycosylation or phosphorylation.
• continuous sequence and “fragments” as used below refer to a linear portion of nucleotides or amino acids derived from a reference sequence, e.g. of the sequences of the present invention as presented in the sequence listing.
• selective hybridization refers to hybridization conditions in which two polynucleotides form duplex nucleotide molecules under stringent hybridization conditions. These conditions are known in the art and e.g. in Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989), ISBN 0-87969-309-6. Examples of stringent hybridization conditions are: (1) hybridization in 4 x SSC at 65 ° C or (2) hybridization in 50% formamide in 4 x SSC at 42 ° C, each followed by several washing steps in 0.1 x SSC at 65 ° C (for 1 hour).
• viral vector refers to genetically modified viruses or bacteria with which those in SEQ ID NO: 1, 2 or 3 DNA sequences carried out, derivatives derived therefrom, fragments, sequences coding for epitopes or epitope strings in different cells, preferably in antigen-presenting cells such as dendritic cells.
• a bacterial vector can also be suitable for directly expressing a polypeptide encoded by SEQ ID NO: 1, 2 or 3, epitopes derived therefrom or epitope strings.
• One aspect of the present invention relates to a nucleotide sequence as described in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
• PBMC peripheral mononuclear blood cells
• Sequence analysis identified a mosaic structure, suggesting extensive intersubtype recombination between the genomes of the prevalent C and (B ') subtype Thai virus strains of that geographic region.
• a RIP analysis Recombinand Identification Program Analysis
• phylogenetic "bootstrapping” revealed a total of 10 breakpoints (i) in the region coding for gagpol, (ii) in vpr and at the 3 'end of the vpu gene and (iii) in the open Reading frame from nef near.
• Thai (B ') sequences therefore comprise (i) several insertions in the coding region of gagpol (nucleotides 478-620, 1290-1830, 2221-2520, each based on the first nucleotide within the start codon of the Gag upon, the GagPol reading frame ), (ii) 3'-vpr, the complete vpu, the first exons of tat and rev (about 1000 nucleotides starting at about nucleotide 138 based on the start codon of the Vpr reading frame) and (iii) the 5'-half of the nef Gene (nucleotides 1-300).
• a sequence of HIV-1 which is the most prevalent C-type virus strain within China, as a basis and starting material is advantageous for the development of preventive or therapeutically usable vaccines.
• the necessary consequences for the development of a successful HIV vaccine candidate are (i) detailed knowledge of the respective epidemiological situation and (ii) the availability of a cloned coding sequence that represents the most prevalent virus strain within a geographical region or a certain population.
• sequences form the basis (i) for the rational conception of preventive and therapeutically usable HIV vaccine candidates, (ii) for the development of specific therapeutic agents, such as therapeutically effective Decoy-Ohgonucleotiden and proteins, antisense constructs, ribozymes and transdominant negative mutants (iii) for the development of lentiviral vectors for gene therapy and (iv) the production of reagents that can be used for diagnosis and monitoring of HIV infection as well as the immunological / viral monitoring of the vaccination process.
• therapeutic agents such as therapeutically effective Decoy-Ohgonucleotiden and proteins, antisense constructs, ribozymes and transdominant negative mutants
• iii for the development of lentiviral vectors for gene therapy
• reagents that can be used for diagnosis and monitoring of HIV infection as well as the immunological / viral monitoring of the vaccination process.
• the conformation epitope according to the The present invention consists of at least 3 amino acids, preferably 5 or more amino acids, which are involved in antibody binding.
• Conformational epitopes can also consist of several sections of either a single protein or - in the case of oligomeric complexes such as the trimeric envelope glycoprotein complex - of several sections of different subunits.
• a linear epitope according to the present invention normally varies in length and comprises at least 8 amino acids to about 15 amino acids or more, with a length of 9 to 11 amino acids being preferred especially in the case of MHC class I restricted CTL epitopes.
• the present invention thus further relates to polypeptides encoded by the nucleotide sequence or fragment or derivative of the nucleotide sequence according to SEQ ID NO: 1, 2 or 3.
• the present invention further relates to polypeptides comprising a continuous sequence of at least 8 amino acids derived from the nucleotide sequence or Fragments or derivatives of the nucleotide sequence according to SEQ ID NO: 1, 2 or 3 are encoded.
• the polypeptide according to the invention preferably comprises an antigenic determinant which naturally triggers an immune reaction in infected people.
• Polypeptides comprising an amino acid sequence encoded by the nucleotide sequence according to SEQ ID NO: 2 or 3 or its derivatives and fragments are particularly preferred.
• epitopes comprising a continuous range of 9 to 11 amino acids which are identical between the polypeptides encoded by SEQ ID NO: 1 and an HIV-I LAI reference isolate, or the 2 or less conserved amino acid substitutions within the sequence comprising 9 to 11 amino acids exhibit. Examples of such epitopes are listed in Example 11.
• the polypeptides according to the invention can be used, for example, as vaccines and therapeutic agents or for diagnosis.
• Another aspect of the present invention relates to a polynucleotide according to SEQ ID NO: 1, 2 or 3. Furthermore, the present invention relates to a polynucleotide fragment of the nucleotide sequence according to SEQ ID NO: 1, 2 or 3, or a polynucleotide which has at least one continuous Sequence of nucleotides comprises, which is capable of selective hybridization to the nucleotide sequence, as shown in SEQ ID NO: 1, 2 or 3. The present invention further relates to derivatives of the polynucleotides or polynucleotide fragments according to the invention.
• the polynucleotide or the polynucleotide fragment preferably comprises a continuous sequence of at least 9 nucleotides, preferably of at least 15 nucleotides, more preferably at least 27 nucleotides, or a longer sequence.
• the polynucleotide or the polynucleotide fragment can also comprise the coding region of the individual HIV genes, such as, for example, from gag, pol, env. Examples are given in SEQ ED NO: 2 and SEQ ID NO: 3.
• Another aspect of the present invention relates to a polynucleotide, comprising at least 2 polynucleotide fragments according to the invention, the sequences of the polynucleotide fragments also overlapping or being separated from one another by a nucleotide placeholder.
• the sequences of the polynucleotide fragments can be identical or different.
• the polynucleotides or polynucleotide fragments according to the invention can be used as vaccines or therapeutic agents or for diagnosis.
• the coding sequence of clone 97cn54 and derivatives thereof, carried out in the form of SEQ ID NO: 1, as representatives of HIV-1 of subtype C can be used as the basis for the following applications:
• subtype C-specific HIV-1 vaccines for prophylactic and therapeutic purposes.
• These subtype-specific vaccines can be used worldwide in all geographic regions where the subtype C virus plays an essential role in the HIV epidemic, e.g. in Latin America, Africa and Asia.
• HIV vaccines to be tested and developed in Southeast Asia and China should be based on the described coding sequence of 97cn54 in order to induce subtype-specific humoral and cell-mediated immune responses.
• HIV-1 subtype C-specific vaccines can be used as a component in a cocktail vaccine, which either takes into account all or a defined selection of the HIV subtypes relevant worldwide.
• the antigens or coding sequences to be delivered to the immune system preferably contain (i) short continuous sections of at least 3 to about 5 amino acids in length or longer sections derived from one of the open ones Reading frames as shown in Table 3, (ii) regions of preferably 9 to 11 amino acids, (iii) combinations of these regions, either separately or as a polypeptide chain (epitope Strings) can be administered, the epitope strings or their amino acid sequences either overlap or separated by amino acids or other placeholders, and particularly preferably complete proteins or the corresponding coding sequences or their variants, which can also include extensive deletions.
• polypeptides which are encoded by the nucleotide sequences or fragments of the nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
• the polypeptide preferably comprises a continuous sequence of at least 8 amino acids, preferably at least 9 to 11 amino acids, particularly preferably at least 15 amino acids or longer sequences or discontinuous epitopes, which are preferably composed of at least three amino acids of a single polypeptide chain or, in the case of oligomeric protein complexes, also put together different polypeptide chains.
• Vaccine constructs based on the coding sequence of 97cn54 include all forms of antigen known in the art and rely on relevant delivery systems.
• Short epitopes encoded by fragments of the nucleic acid sequences according to SEQ ID NO: 1 to 3, and each comprising three to five amino acids, preferably comprising 9 to 11 or more amino acids, can preferably be produced synthetically.
• Such peptides contain either a B cell epitope, an MHC class ⁇ -restricted T helper epitope, an MHC class I restricted cytotoxic T cell epitope or combinations of the variants mentioned.
• Individual epitopes can overlap or be separated from one another by placeholders, preferably consisting of glycine and / or serine residues.
• branched-chain peptides can be produced either during the synthesis or with the aid of the customary and commercially available homo- and heterobifunctional chemical crosslinking agents after the synthesis and purification of the corresponding peptides.
• peptides which are not very immunogenic per se can also be conjugated to selected carrier proteins such as ovalbumin by crosslinking, inserted into carrier proteins by genetic engineering or fused to their N or C terminus.
• carrier proteins are preferably (i) when expressed in suitable cell culture systems (see below) or (ii) after suitable refolding of the purified, denatured protein, are able to form particulate structures in which B cell epitopes preferably lie on the surface of the particulate carrier come.
• HBV hepatitis B virus
• HBV surface protein HBV surface protein
• HIV group-specific antigen the polyomavirus VP1 protein
• Pappillomvirus Ll protein the Pappillomvirus Ll protein or the TyA protein of yeast
• VLP virus-like particles
• Epitope strings and polypeptides encoded by fragments of the nucleic acid sequences according to SEQ ID NO: 1 to 3, with a length greater than 30, preferably greater than 50 amino acids, as well as polypeptides with a tendency to form particulate structures (VLP) can, according to the prior art, be used in prokaryotes produced and cleaned.
• Such plasmids accordingly contain a bacterial origin of replication, e.g. ColEl, usually a selection marker such as resistance to kanamycin or ampicillin, a constitutively active or inducible transcription control unit such as, for example, the LacZ or Tac promoter, and the signals for translation start and stop.
• cleavable fusion fractions and cleaning aids such as, for example, glutathione-S-transferase or cleaning aids such as e.g. Oligohistidine-t ⁇ gs (catchers) can be used.
• the DNA or RNA sequences which (i) are used to produce the epitope strmgs, complete proteins or virus-like structures in eukaryotic cell cultures such as yeast cells, fungi, insect cells or mammalian cells or (ii) for the direct administration of DNA used for immunization purposes can rely on the use of the codons as used by the virus itself.
• the use of the codons wherever technically possible, can be adapted to the most or second most frequently used codons in genes which are highly expressed in the respective production system. Examples of the optimization of the codon use in a polygen optimized from a safety point of view, including the genes Gag, Pol and Nef, and in the coat protein gene are given in SEQ ID NO: 2 and SEQ ID NO: 3.
• SEQ IDs NO: 2 and 3 are specified in Example 15.
• the establishment of cell lines for the production of epitope strings, polypeptides or virus-like structures in the cell culture systems mentioned can, based on the prior art, be based on vectors which, in addition to a bacterial origin of replication, a positive or negative selection marker, and above all the corresponding control regions for compliance with the rules May include transcription and translation of the foreign protein.
• the components of the DNA vaccine constructs described below are also examples of the modules that can also be found in vectors for the expression of the epitope strings, polypeptides or complete proteins in different mammalian cell cultures.
• the simplest form of immunization is the direct administration of a pure DNA vaccine.
• This contains a transcription control region, also called a promoter / enhancer region, on the 5 'side of the coding region, which can optionally be followed by a functional intron to increase gene expression, (ii) a Kozak sequence including a translation start codon and am 3 'end of the foreign gene is a translation termination codon followed by a polyadenylation signal sequence.
• the promoter / enhancer region can preferentially support constitutive expression of the desired gene product and is, for example, from the transcriptional control region of an immediately early (IE) cytomegalovirus gene (CMV-IE) or the Rous-Sarcoma virus (RSV) LTR (long terminal repeat) derived.
• IE immediately early
• CMV-IE cytomegalovirus gene
• RSV Rous-Sarcoma virus
• an inducible form of a transcription control region such as a Tet on / Tet o promoter can also be used, in which the transcription is regulated, for example, by the administration of tetracycline or corresponding analogs.
• a transcription control region such as a Tet on / Tet o promoter
• the transcription is regulated, for example, by the administration of tetracycline or corresponding analogs.
• cell type-specific regulated transcription control regions is possible, such as the upstream of the muscle creatine kinase gene (MCK gene; muscle-specific expression), the CD4 receptor gene or the MHC class II genes (preferential expression in antigen presenting cells) located promoter / enhancer regions.
• chimeric combinations of (i) cell type specific promoters and (ii) viral enhancer regions are also used to combine the advantages of tissue-specific expression with those of the strong transcriptional activity of viral enhancers.
• the enhancement of gene expression by incorporating a functional intron, which is generally located 5 'to the side of the open reading frame, is increased Core export rate is spliced back in comparison to unspliced transcripts and is achieved, for example, by inserting an intron located in the ⁇ -globin gene.
• a preferred form of a DNA vaccine based on SEQ ID NO: 1, 2 or 3 additionally contains a replicon derived from alpha viruses such as, for example, Semliki Forest or Venezuela encephalitis viruses (SFV, VEE).
• SFV Semliki Forest or Venezuela encephalitis viruses
• the nuclear transcription control unit described above and the optionally considered intron are first followed by the region coding for the VEE or SFV non-structural proteins (NS).
• NS non-structural proteins
• the actual foreign gene follows only on its 3 'side, the cytoplasmic transcription of which in turn is regulated by an NS-sensitive promoter. Accordingly, starting from the nuclear transcription control unit, a long transcript is generated over several open reading frames, which is then translocated into the cytoplasm.
• NS proteins synthesized there then activate the cytoplasmic transcription of the foreign genes by binding to the corresponding control region.
• This amplification effect usually leads to an abundant RNA synthesis and consequently high foreign protein synthesis rates.
• the latter in direct comparison with conventional plasmids which do not have the effect described by cytoplasmic RNA amplification, generally allows a significant reduction in the amount of plasmid to be administered with at least comparable immunogenicity.
• the peptides, proteins, virus-like particles and DNA constructs described above can be administered by intramuscular, subcutaneous, intradermal, intravenous injection, the prior art being used in each case for the administration of the proteinaceous antigens.
• DNA immunization either conventional syringes with injection needles can be used, or equipment that does not need needles and can usually introduce the DNA directly into the desired tissue using compressed air.
• this also includes the intranasal and oral application of DNA-containing vaccine formulations by spray-like devices.
• the DNA can also be attached to solid supports such as e.g. Gold beads are conjugated and administered into the corresponding tissues, for example under air pressure.
• the aforementioned proteinaceous antigens and DNA constructs can also be used with adjuvants, usually stimulators of the Immune response, combined or administered in a sequential order with the adjuvants.
• adjuvants usually stimulators of the Immune response
• Conventional adjuvants such as aluminum hydroxide or aluminum hydroxyphosphate result in a stimulation of the humoral immune response, which is also characterized by high antibody titers of the IgGl subtype.
• CpG oligonucleotides consistensus nucleus motif: purine-purine-CpG-pyrimidine-pyrimidine
• chemically modified derivatives thereof phosphothioate oligonucleotides; oligonucleotides with a peptide backbone
• Thl Immunity characterized by high antibody titers of the IgG2a subtype and the induction of Thl cytokines such as ⁇ -IFN, IL-2 and EL-12.
• Biodegradable particles include, for example, PLA (L-lactic acid), PGA (polyglycolic) or PLGA [poly (D, L-lactide-co-glycolide)] microspheres or derivatives thereof, cationic microparticles or carrier substances derived from bacterial capsule polysaccharides.
• ISCOMS immunostimulating complexes, which originate from water-soluble extracts of the bark of Quillaja saponaria and were further purified by means of chromatographic processes.
• ISCOMS immunostimulating complexes, which originate from water-soluble extracts of the bark of Quillaja saponaria and were further purified by means of chromatographic processes.
• a detailed overview of the various adjuvants and administration aids corresponding to the state of the art can be found at http://www.niaid.nih.gov/aidsvaccine/pdf/compendium.pdf [Vogel, FR, Powell, MF and Alving, CR " A Compendium of Vaccine Adjuvants and Excipients (2nd Edition)].
• viral and, alternatively, bacterial vectors can be used for the convenient presentation of epitope-Str gs-, polypeptides and virus-like particles.
• genetically modified Salmonella and Listeria are particularly suitable due to their natural cell tropism, DNA vaccine constructs in antigen-presenting cells such as monocytes, macrophages and especially in dendritic cells.
• the genetic engineering changes can, among other things, help the DNA to reach the cytoplasm of the antigen-presenting cell without damage.
• a DNA vaccine construct enters the cell nucleus, where the corresponding reading frame is transcribed using the cellular resources and proteins via a eukaryotic, preferably viral or cell type-specific promoter. After the RNA has been transported into the cytoplasm, the corresponding gene product is translated and, depending on its nature, modified post-translationally and assigned to the corresponding cellular compartment.
• Bacterial vectors can also be used to induce mucosal immunity, preferably after oral administration.
• the corresponding antigens are produced by the bacterial transcription and translation machinery and are therefore not subject to the post-translational modifications that are otherwise common in mammalian cells (no corresponding Glycosylation; no secretory pathway).
• viral vectors can also be used directly for immunization. This can be done either ex vivo, for example for the infection of antigen-presenting cells, which are then administered to the vaccine, or directly in vivo by subcutaneous, intradermal, intracutaneous, intramuscular or intranasal immunization with the recombinant virus, which is a favorable antigen Presentation with appropriate immunization success can be achieved.
• vaccinia viruses such as the modified vaccinia virus Ancara (MVA) attenuated by passage through chicken cells, the genetically attenuated vaccinia strain New York ( ⁇ YVAC) or the avian vaccinia viruses (fowlpox, canarypox) endemic in birds and cell-mediated immune responses can be induced in the vaccinated subjects.
• vaccinia viruses such as the modified vaccinia virus Ancara (MVA) attenuated by passage through chicken cells, the genetically attenuated vaccinia strain New York ( ⁇ YVAC) or the avian vaccinia viruses (fowlpox, canarypox) endemic in birds and cell-mediated immune responses can be induced in the vaccinated subjects.
• avian vaccinia viruses endemic in birds and cell-mediated immune responses can be induced in the vaccinated subjects.
• recombinant alpha viruses including the Semliki Forest virus or the Venezuela ence
• Attenuated HIV viruses can also be generated on the basis of SEQ ID NO: 1, 2, or 3 and used for immunization purposes, provided that the regulatory sequences (LTR, long terminal repeat) that flank the coding region are used by cloning methods according to the prior art , can be added. Adequate attenuation of the virus can then be achieved according to the prior art by one or more deletions, for example in the Nef gene
• nucleic acid sequences carried out in the examples SEQ ID NO: 1 and SEQ ID NO: 3 as well as peptides, proteins or virus-like particles derived therefrom can also be used as components of viral vectors for gene transfer.
• the polypeptides encoded by the GagPol gene can encode the packaging and receptor functions of e.g. Provide lenti or retroviral vectors.
• suitable plasmid vectors which support the simultaneous expression of the GagPol and VSV-G (vesicular stomatitis virus coat protein G) gene and ensure the packaging of a therapeutic transgene
• virus particles are generated which are also capable of resting, postmitotic or transduce end differentiated cells. This method of generating transduction-competent virus particles can be made considerably easier and more efficient, for example by establishing stable cell lines, e.g.
• adenoviruses based on human embryonic kidney cells (HEK293), which express the GagPol polyprotein constitutively or under the control of an inducible promoter.
• HEK293 human embryonic kidney cells
• recombinant adenoviruses can also be generated, which encode the packaging functions, the receptor functions and the transgene functions or combinations thereof, and thus serve as a tool for the ex vivo, in situ and in vivo delivery of retro- or lentiviral vectors.
• the coat proteins or derivatives encoded by SEQ ID NO: 3 can provide the receptor function for lenti, spuma or retroviral vectors or other vectors based on coated viruses by incorporation into the lipid bilayer.
• packaging lines can also be generated, for example, in which the GagPol proteins from retro, spuma and preferably lentiviruses, as well as those from SEQ ID NO: 1 and 3 derived coat proteins can be expressed either constitutively or under the control of an inducible, optionally an activity-regulable promoter.
• chimeric viruses can be generated which, in addition to the natural coat protein or instead of the natural coat protein, have a SEQ ID NO: 1 or SEQ ID NO: 3 derived coat protein on the surface.
• proteins or virus-like particles derived from SEQ IDs NO: 1 to 3 (i) polyclonal antisera, (ii) monoclonal antibodies (mouse, human, camel), (iii) antibody derivatives such as single-chain Antibodies, humanized antibodies, bispecific antibodies, phage antibody banks or (iv) other highly affinity binding polypeptides such as, for example Derivatives of the hPSTI (human pancreatic secretory trypsin inhibitor) are generated.
• hPSTI human pancreatic secretory trypsin inhibitor
• the peptides, proteins or nucleic acid sequences derived from SEQ ID NO: 1, 2 or 3 can be used for diagnostic purposes, e.g. for serodiagnostics and for the application of nucleic acid hybridization techniques or nucleic acid amplification systems or combinations thereof.
• the polynucleotide fragments of the nucleotide sequence according to SEQ ID NO: 1 can preferably be used in a polymerase chain reaction.
• the polynucleotide fragments of the nucleotide sequence according to SEQ ID NO: 1 are particularly preferably used for diagnosis by means of DNA chip technology.
• PBMC Peripheral blood mononuclear cells
• PHA phytohaemaglutinin
• Proviral DNA was extracted from productively infected PBMCs from more than one hundred selected HIV-1 positive IDUs from the northwestern provinces of China (Qiagen Inc., Valencia, CA).
• the nested V R was used to amplify the coding region for env C2V3.
• the PCR products were described using the T ⁇ ⁇ -cvc / e method using fluorescent dye-labeled terminators (Applied Biosystems, 373A, Foster City, CA) as described recently (Bai et al. 1997; Yu et al. 1997 ) sequenced directly. Multiple sequence comparisons were carried out using the Wisconsin software package Genetics Computer Group with the correction methods according to Kimura (GCG, 1997, version 9).
• Example 3 Phylogenetic family tree analyzes were performed on all sequences obtained using the PHYLIP software package. Evolutionary distances were calculated using the maximum parsimony method and indicated by the cumulative horizontal length of the branches. The statistical robustness of the neighbor joining family tree was checked by bootstrap resampling as recently described (Graf et al. 1998).
• both 97cn54 and cn-con-c show amino acid deviations at positions 13 (H—> R) and 19 (A—> T), both of which are characteristic of isolates of the subtype C are (C_consensus).
• Table 1 The amino acid sequence comparison of the V3 loops of consensus sequences of different subtypes of HIV-1 (A-O) and selected isolates of subtype C from different countries.
• the overall V3 consensus sequence was determined by comparing the consensus sequences of different subtypes (A-O).
• cn-con-V3 represents the consensus sequence of HIV-1 strains of subtype C that are prevalent in China.
• 97cn54 was selected as the representative standard isolate of the prevalent HIV-1 strains of subtype C that are found in China.
• "-" means no exchange for the V3 consensus sequence, lower case letters mean an amino acid substitution and ".” means gaps. All consensus and isolate sequences for multiple comparisons were obtained from the Los Alamos database.
• Example 6 The sequence coding for the 97cn54 coat protein is most closely related to type C virus strains from India. Phylogenetic family tree analysis, originally based on the C2V3 sequences of the env gene, revealed that both 97cn54 and the consensus sequence of the Chinese isolates of subtype C are compatible with the strains of subtype C from India (ind8, dl024, c-93in905, c-93in999, c-93inl l246) Africa (c-eth2220, c-ug286a2), and from South America (92br025, nof, cam20 and sml45) group (cluster).
• HIV-1 genomes were amplified using the Expand Long Template PCA system (Boehringer-Mannheim, Mannheim, Germany) as described by Graf et al. (1998) and Salminen et al. (1995).
• the primers were positioned in conserved regions within the long terminal repeats (LTR) of HIV-1: TBS-AI (5'-ATC TCT AGC AGT GGC GGC CGA A) and NP-6 (5'-GCA CTC AAG GCA AGC TTT ATT G).
• LTR long terminal repeats
• Purified PCR fragments were blunt-ended in a SrCR digested pCR script vector (Stratagene, Heidelberg, Germany) and transformed into the E. co // strain DH5 ⁇ .
• RFLP restriction fragment length polymorphism
• Example 8 DNA sequences were assembled using Lasergene software (DNASTAR, Inc., Madison, WI) on Macintosh computers. All reference sequences of the subtypes of this study are from the Los Alamos HIV database. Similarities in the nucleotide sequence were calculated using the Smith and Waterman local homology algorithm. Multiple sequence comparisons with available sequence data from other subtypes were carried out using the Wisconsin software package Genetics Computer Group (GCG, 1997, version 9).
• the 9078 bp genomic sequence of isolate 97cn54 contained all known structural and regulatory genes of the HIV-1 genome. No significant deletions, insertions or rearrangements were found. The similarities in the nucleotide sequence were examined by comparing all coding sequences (CDS) of 97cn54 with consensus sequences of different genotypes and selected subtype isolates (Table 2). The highest homologies of the reading frames of gag, pol, env and vif to the corresponding consensus sequences of subtype C ranged from 93.93 to 95.06%. This observation significantly expanded the sequence comparison described above and the phylogenetic family tree analysis based on C2V3 (see Table 1 and Figure 1).
• the virus isolate selected belongs to the group of recently published virus strains of subtype C.
• the homology values determined by this type of analysis for the genes tat, vpu, vpr and nef were not sufficient to allow a clear assignment of these reading frames to virus strains of subtype B or C (Table 2).
• the highest homologies to subtype B were registered (94.24% o), while the homology to the consensus sequence of subtype C was only 78.23%.
• Table 2 Nucleotide sequence comparison of all coding sequences (CDS) between 97cn54 and DNA sequences containing either (1) consensus sequences of certain HIV-1 subtypes (obtained from the Los Alamos HIV database) or (2) isolates of the standard -Subtypes C (92br025 and eth2220) and B (mn and rl42) represent.
• the data indicate the identity of a specific sequence with 97cn54 in percent.
• Non-unique nucleotide positions within the consensus sequences were rated as identical. The highest homologies are highlighted in bold. 7 "means that no consensus sequence was available from the Los Alamos database.
• the recombinant identification program (RIP, version 1.3; http://hiv-wew.lanl.gov/tools) was used to identify potential mosaic structures within the overall sequence of this clone (window size: 200; threshold for statistical significance: 90 %; Dealing with gaps: STRIP; informative mode: OFF). Gaps have been introduced around the Enable comparison.
• the background sequences of the subtypes in this analysis were: u455 (subtype A), RL42 (Chinese subtype B-Thai (B ')), eth2220 (subtype C), z2d2 (subtype D), 93th2 (subtype A / E).
• Example 12 Intersubtype recombination in the env coding region of 97cn54
• 97cn54 is a C / (B ') intersubtype mosaic virus with 10 breaks of intersubtype recombination that is most prevalent among IDUs within the northwestern provinces of China.
• a schematic representation of the (BVC) mosaic genome of isolate 97cn545 is shown in FIG. 6.
• To preserve beyond subtypes To estimate cross-reactive CTL epitopes, the predicted protein sequences of 97cn54 were compared with the known and best mapped LAI-specific CTL epitopes.
• 75, 55, 40 and 24 are in Gag, (pl7, p24, pl5), in reverse transcriptase (RT), in g ⁇ l20 and in g ⁇ 41, respectively.
• the numbers refer to the 5 'end of the DNA sequence shown in SEQ ID NO: 1.
• C-gpnef The gene C-gpnef was cloned into the only Kpnl / Sacl restriction sites of the pCR-Script amp (+) cloning vector (Stratagene).
• the N-terminal glycine was exchanged for alanine (nucleotide sequence GGC) in order to avoid targeting of the polypeptide to the cytoplasmic membrane and the subsequent secretion of assembled virus-like particles via budding.
• Example 16 The GagPolNef polygen encoded by SEQ ID NO: 1 was inserted into the vector pcDNA3.1 via Kpnl / Xhol and transformed into the E.coli strain XLlblue.
• the ability of the GagPolNef expression vector to induce a Gag-specific antibody response was analyzed in female BALB / c mice (Fig. 9). Two groups of 5 animals each received an intramuscular (im) primary immunization of 100 ⁇ g DNA per immunization, followed by 2 subsequent immunizations 3 and 6 weeks later (group 1: pcDNA-GagPolNef; group 2: pcDNA). A control group (group 3) was only immunized with PBS.
• the total titers of Gag-specific IgG were determined against purified Gag protein in an ELISA.
• Vaccination with pcDNA-GagPolNef resulted in a rapid induction of high titers of Gag-specific antibodies (1: 4,000), which was characterized by a typical Thl profile of antibody isotypes (IgG2a »IgGl).
• the two control groups 2 and 3 provided no evidence of the generation of Gag-specific antibodies.
• Antibody titers increased almost 100-fold (1: 20,000) 1 week after the first follow-up immunization and reached gag-specific endpoint titers of 1: 80,000 one week after the second booster immunization. At no time could a significant, Gag-specific antibody response be detected in the two control groups.
• Example 17 Example 17:
• Antigen-specific cytokine secretion as an indication of the induction of a T helper memory response was analyzed from spleen cells, which were taken 5 days after the second follow-up immunization.
• a comparatively reduced gIFN production was observed in spleen cells which were obtained from mice after three subcutaneous (s.c.) or intradermal (i.d.) immunization with pcDNA-GagPolNef according to the same scheme as above.
• DNA vaccine IL-4 (pg / ml) IL-5 (pg / ml) JJFN- ⁇ (pg / ml) pcDNA-GagPolNef ⁇ 8 ⁇ 16 3220 ⁇ 840
• Example 18 To test the ability of pcDNA-GagPolNef to induce Gag-specific CTLs, spleen cells were tested in vitro 3 weeks after primary immunization with pcDNA-GagPolNef (group 1), pcDNA (group 2) and PBS (group 3) mixed-lymphocyte-tumor cell culture specifically restimulated for 6 days and then examined for their cytotoxic activity.
• Gag protein subtype B virus (IHB isolate) derived AMQMLKETI peptide (single letter code), which was used in this experiment for in vitro restimulation as well as for the determination of the specific cytotoxic activity, is known to represent a D d restricted CTL epitope in the BALB / c mouse.
• G ⁇ g-specific cytotoxic T cells were found after a single injection with the pcDNA-GagPolNef plasmid, but not in one of the two control groups 2 and 3. The treatment of spleen cells with the above-mentioned peptide did not result in an in vitro priming Gag-specific cytotoxic T cell.
• McCutchan, F.E. (1998). Filling genome sequences of human immunodeficiency virus type 1 subtypes G and A G intersubtype recombinants. Virology 247, 22-31. Esparza, J., Osmanov, S., and Heyward, W.L. (1995). HIV preventive vaccines. Progress to date.
• the heterosexual human immunodeficiency virus type 1 epidemic in Thailand is caused by an intersubtype (A / E) recombinant of African origin. J Virol. 70, 7013-7029.
• HIV-1 World Health Organization Network for HIV Isolation and Characterization (1994). HIV-1

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